This proposal explores the mechanism of control of eukaryotic transcription and the role of phosphorylation as a modulator of RNA synthesis in higher organisms and dividing cells in culture. Specifically, it is proposed that endogenous nuclear protein kinases are involved in transcription at the level of the DNA-dependent RNA polymerases (nucleolar form I, nucleoplasmic forms II and III). That is, RNA polymerases are proposed to be phosphorylated (activated) at some stage in transcription (initiation) and dephosphorylated upon termination of RNA synthesis. Experiments are proposed to purify the nuclear protein kinases and acid phosphatases to begin to characterize their possible role in the control of ribosomal, messenger, and transfer RNA transcription by the respective forms I, II, and III RNA polymerase and study their regulatory modulation relative to changes in RNA polymerase activity. Isolated nucleoli will be employed as a model system to purify nuclear phosphatase(s) which may be specific for the release of chromatin bound RNA polymerase I. Using endogenous nuclear protein kinase, the purified enzyme will be rephosphorylated to determine any different kinetic properties from the dephospho enzyme. Similiar studies will be performed with RNA polymerases II and III to determine the effect of phosphorylation on the kinetic properties of these enzymes. RNA polymerases, protein kinase, and acid phosphatase activities will also be measured throughout the cell cycle of CHO cells induced to synchrony by isoleucine deficiency. RNA polymerases will be assayed in both nuclei and on DEAE Sephadex columns to determine endogenous versus total RNA polymerase activities. These studies are designed to provide knowledge of eukaryotic transcription, how it is modulated and controlled, and the role of additional enzymes and/or factors in these control mechanisms.